The identification of low molecular weight analytes (LMWRs) in biological fluids is a rather complex analytical task both in terms of selectivity and detection limits. The most practical competitive assays require selective receptors (e.g. antibodies) against the analyte. Since LMWR themselves do not cause an immune response, conjugates of LMWR with a protein are used to obtain antibodies. As shown in this work, the method of creating such a conjugate as well orientation of immobilized analyte’s analog has a dramatic effect on the efficiency of analyte detection. We demonstrate the validity of this requirement using the example of SPR detection of 17β-Estradiol (E2), one of the most important steroid hormones widely spread in environments. An immunospecific detection of E2 following the competitive inhibition format uses E2 analog directly immobilized on the gold surface through the specific spacer providing both optimal detection conditions, stability of interfacial architecture and low level of non-specific sorption. Comprehensive studies have shown that the efficiency of the system strongly depends on which fragment of E2 is attached to the surface. If antibodies obtained against conjugates with E2 bound in the 17th position of the steroid ring were used, then the antibodies did not recognize the immobilized E2 analogue. However, serum obtained using a conjugate where the BSA-carrier protein is attached to the 3rd position of E2 instead of the 17th showed a high affinity for surface-bound E2. Using an illustrative example of E2 detection, we conduct a comparative analysis of the elements of success and failure of the LMWR analytical analysis in terms of the spatial structure of sensitive zones and various biochemical information contained in certain fragments of steroid hormones important for the formation of unique antibody recognition centers.